An integrated map of the murine hippocampal proteome based upon five mouse strains

With the advent of proteomics technologies it is possible to simultaneously demonstrate the expression of hundreds of proteins. The information offered by proteomics provides context-based understanding of cellular protein networks and has been proven to be a valuable approach in neuroscience studies. The mouse hippocampus has been a major target of analysis in the search for molecular correlates to neuronal information storage. Although human and rat hippocampal samples have been successfully subjected to proteomic profiling, no elaborate analysis providing the fundamental experimental basis for protein-expression studies in the mouse hippocampus has been carried out as yet. This led us to construct a master map generated from the individual hippocampal proteomes of five different mouse strains. A proteomic approach, based upon 2-DE coupled to MS (MALDI-TOF/TOF) has been chosen in an attempt to establish a comprehensive reference database of proteins expressed in the mouse hippocampus. 469 individual proteins, represented by 1156 spots displaying various functional states of the respective gene products were identified. Proteomic profiling of the hippocampus, a brain region with a pivotal role for neuronal information processing and storage may provide insight into the characteristics of proteins serving this highly sophisticated function.     

Proteomics as a tool for examining the toxicity of heavy metals

Examining the toxic effects of heavy metals on protein expression can be useful for gaining insight into the biomolecular mechanisms of toxicity and for identifying potential candidate metal-specific protein markers of exposure and response. In this article, we present the state of the art of proteomics in metal-toxicity-related studies. We consider different methods used for sample preparation that depend on the nature of the sample (plants, microorganisms and animals). We also describe different proteomic strategies, both gel-based and gel-free technologies, including two-dimensional gel electrophoresis (2-DE) and multi-dimensional protein-identification technology (MudPIT). We critically review the advantages and the disadvantages of such techniques and discuss the main studies carried out so far. We also comment on future applications and potential research interests within this field.     

Dialysis-assisted two-dimensional gel electrophoresis

2-DE is an important tool in proteomics research. However, intrinsic gel-to-gel variability of 2-DE often masks the biological differences between the samples and compromises quantitative comparison of protein expression levels. Here, we describe a modification of 2-DE that results in improved matching and quantification of proteins. This was accomplished by performing IEF of two samples in two IPG strips separated by a dialysis membrane. After IEF running, the strips were separated and the SDS-PAGE dimension was accomplished on two individual gels. After gel staining with CBB, ImageMaster 2D Platinum software (Amersham) was used for spot detection and quantification. Analysis of protein extracts from C2C12 myoblasts by this method resulted in 99% spot-matching efficiency and CV in stain intensity (% volume) was less than 0.5 for 98% of spots. We conclude that this technique, called dialysis-assisted gel electrophoresis, gives superior spot matching and quantitative reproducibility compared to IEF conducted on separate strips.     

From proteomics to genomics

Presently, science is moving from genomics to proteomics in order to get insight into the functional network of gene expression. Actually however, proteomics is much older than genomics and dates back to the introduction of the two-dimensional gel electrophoresis technique (2-DE) independently by Klose and O'Farrell. Based on this approach almost all cellular proteins can be separated. New developments in mass spectrometry allowed identification of single spots in the 2-DE protein pattern, including the underlying genes. Joachim Klose has focused his pioneering 2-DE studies on mouse models with special emphasis on quantitative protein variants. According to him, proteins are living molecules exhibiting a characteristic protein phenotype.     

Trends in sample preparation for classical and second generation proteomics

Sample preparation is a fundamental step in the proteomics workflow. However, it is not easy to find compiled information updating this subject. In this paper, the strategies and protocols for protein extraction and identification, following either classical or second generation proteomics methodologies, are reviewed. Procedures for: tissue disruption, cell lysis, sample pre-fractionation, protein separation by 2-DE, protein digestion, mass spectrometry analysis, multidimensional peptide separations and quantification of protein expression level are described.     

Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels

Progress in the field of proteomics is dependent upon an ability to visualise close to an entire protein complement via a given array technology. These efforts have previously centred upon two-dimensional gel electrophoresis in association with immobilised pH gradients in the first dimension. However, limitations in this technology, including the inability to separate hydrophobic, basic, and low copy number proteins have hindered the analysis of complete proteomes. The challenge is now to overcome these limitations through access to new technology and improvements in existing methodologies. Proteomics can no longer be equated with a single two-dimensional electrophoresis gel. Greater information can be obtained using targeted biological approaches based upon sample prefractionation into specific cellular compartments to determine protein location, while novel immobilised pH gradients spanning single pH units can be used to display poorly abundant proteins due to their increased resolving power and loading capacity. In this study, we show the effectiveness of a combined use of two differential subproteomes (as defined by relative solubilities, cellular location and narrow-range immobilised pH gradients) to increase the resolution of proteins contained on two-dimensional gels. We also present new results confirming that this method is capable of displaying up to a further 45% of a given microbial proteome. Subproteomics, utilising up to 40 two-dimensional gels per sample will become a powerful tool for near-to-total proteome analysis in the postgenome era. Furthermore, this new approach can direct biological focus towards molecules of specific interest within complex cells and thus simplify efforts in discovery-based proteome research.     

Application of thiophilic chromatography to deplete serum immunoglobulins in sample preparation for bidimensional electrophoresis

Serum is a typical sample for non-invasive studies in clinical research. Its proteome characterization is challenging, since requires extensive protein depletion. Methods used nowadays for removal of high-abundance proteins are expensive or show quite often a low loading capacity, which has strong repercussions on the number of samples and replicates per analysis.In order to deplete immunoglobulins (Igs) and albumin (HSA) from 1 mL serum samples, we have developed a protocol based on a combination of thiophilic chromatography, not previously used in clinical proteomics, and a HSA-specific resin. Ig/HSA-depleted samples, immunoglobulinome and albuminone were analyzed by 2-DE. Thiophilic chromatography, coupled with HSA-depletion, allows a good 2-DE resolution as well as the visualization of new spots. Moreover, it yields enough protein to evaluate technical variability and facilitate subsequent protein identification. To validate the protocol, we carried out a preliminary comparative study between triplicate Igs/HSA-depleted serum samples from healthy control individuals and recently diagnosed/untreated rheumatoid arthritis (RA) patients. RA patients showed several acute phase proteins, as well as additional serum proteins, differentially and significantly regulated.Therefore, thiophilic chromatography can be used as an efficient and economical method in 2-DE to deplete immunoglobulins from large human serum samples before a more extensive fractioning.     

Mass spectrometry meets chip technology: a new proteomic tool in cancer research?

DNA chip technologies are the most exiting genomic tools, which were developed within the last few years. It is, however, evident that knowledge of the gene sequence or the quantity of gene expression is not sufficient to predict the biological nature and function of a protein. This can be particularly important in cancer research where post-translational modifications of a protein can specifically contribute to the disease. To address this problem, several proteomic tools have been developed. Currently the most widely used proteomic tool is two-dimensional protein gel electrophoresis (2-DE), which can display protein expression patterns to a high degree of resolution. As an alternative to 2-DE, a preliminary study using a new technique was employed to generate protein expression patterns from whole tissue extracts. Surface-enhanced laser desorption/ionization (SELDI) allows the retention of proteins on a solid-phase chromatographic surface (ProteinChip Array) with direct detection of retained proteins by time of flight-mass spectrometry (TOF-MS). Using this system, we analyzed eight cases of renal cell carcinoma (RCC) including normal, peripheral and central tumor tissue as well as four microdissected cases of cervical intraepithelial neoplasia (CIN) and three microdissected cases of cervix uteri carcinoma. Differentially expressed proteins were found by comparing the protein expression patterns generated using SELDI-based TOF-MS of tumor tissue with normal and neoplastic tissue, respectively. By applying this fast and powerful ProteinChip array technology it becomes possible to investigate complex changes at the protein level in cancer associated with tumor development and progression.     

A comparison of the consistency of proteome quantitation using two-dimensional electrophoresis and shotgun isobaric tagging in Escherichia coli cells

An important consideration in the measurement of quantitative changes in protein expression is the consistency of the observations for a given technique as well as the reproducibility of the experiment. A quantitative assessment of the technical and biological variability is crucial to avoid erroneous inferences and conclusions. Two methods for measuring quantitative changes in protein expression are two-dimensional electrophoresis (2-DE) and shotgun proteomics of isobaric-tagged samples using iTRAQ reagents. An assessment of changes in Escherichia coli protein expression in response to rhsA induction demonstrates that half of the quantified protein expression ratios have a coefficent of variation (CV) less than 0.31 using 2-DE and less than 0.24 using isobaric tags; whereas 95% of the quantified protein expression ratios have a CV less than 0.81 using 2-DE and less than 0.53 using isobaric tags. The selective removal of outlier data points from the shotgun method using Grubb's and Rosner's statistical outlier tests improves the consistency of the quantitation data obtained.     

The future of post-genomic biology at the proteomic level: an outlook

Drug discovery and early-stage drugs and biomarkers development is a continuous adaptation and maturation process. The cycle of changes based on new findings is coupled with shifts in research priorities and make this part of pharmaceutical research a challenging endeavour. Over the last years, the emphasis on genomics has shifted to proteomics, the science of understanding how proteins translate gene information into function, and metabonomics, the science of small metabolites that are further apart from genomic projects. Proteomics describes the analysis of the protein complement of a biological sample with respect to temporal and spatial resolution. This technology is based on separation of complex protein mixtures by 2D gel-electrophoresis, in gel digest and mass spectrometric analysis of the protein fragments. Proteomics has been recently flanked by peptidomics, a new research direction aimed at the comprehensive analysis of small (1-20 kDa) polypeptides, thus covering the gap between proteomics and metabonomics. The refinement of peptidomics is based on an essential paradigm related to modularity and diversity. Peptides are a paramount example of how one single gene can release multiple functionalities. We can expect fast progress in understanding protein and peptide networks from a systems biology approach ending in the discovery of new peptide targets. However, the way from a complex sample to potential diagnostic and therapeutic targets will depend on technological developments and from the ability to discriminate true disease-related signals from false positive and negative signals, and the way from target discovery to target validation will not be short.     

2-DE proteomic analysis of the model cyanobacterium Anabaena variabilis

Cyanobacteria are photosynthetic bacteria capable of producing hydrogen and secondary metabolites with potential pharmaceutical applications. A limited number of cyanobacterial 2-DE proteomic studies have been published, most of which are based on Synechocystis sp. PCC 6803. Here, we report the use of 2-DE, ESI-MS/MS and protein bioinformatics tools to characterize the proteome of Anabaena variabilis ATCC 29413, a heterocystous nitrogen-fixing cyanobacterium that is a model organism for the study of nitrogen fixation. Using a 2-DE workflow that included the use of a detergent-based extraction buffer and 3-10 nonlinear IPG strips resulted in the identification of 254 unique proteins, with significantly better coverage of basic and low-abundance proteins that has been reported in 2-DE analyses of Synechocystis sp. A set of protein bioinformatics tools was employed to provide estimates of protein localization, hydrophobicity, abundance and other properties. The characteristics of the A. variabilis proteins identified in this study were compared against the theoretical proteome for this organism, and more generally within the cyanobacteria, to identify opportunities for further development of 2-DE-based cyanobacterial proteomics.     

Two dimensional electrophoretic analysis of human tears: collection method in dry eye syndrome

Tear proteomics, by 2-DE, can give a fingerprint of the protein profile, which is well suited in clinical proteomics for biomarker identification and in diagnostics. The mode of tear collection can influence the representation of the proteins in the tear and therefore it is important to use the appropriate method. In this study, capillary and Schirmer mode of tear collection was done in the healthy controls and the Schirmer method was validated in dry eye syndrome conditions. 2-D PAGE of normal and dry eye tear was performed using pH 3-10 linear IPG strips followed by 13% SDS-PAGE. The spot intensity was analyzed by the PD quest software. The two methods were compared using Bland-Altman statistical tool. The 2-D map of capillary and Schirmer tear showed 147 ± 8 spots and 145 ± 7 spots respectively. Both the collection methods were in agreement with each other and were comparable. Dry eye tear protein showed differential expression of proteins as observed in 25-35 kDa region. One of the significantly reduced protein was identified as proline-rich 4 protein. Schirmer method of tear collection is reliable in patients with dry eye, which can display the differential protein expression and help in biomarker identification.     

K-Means聚类分析法筛选柠檬香茅茎叶差异蛋白及鉴定

柠檬香茅含有大量的香茅精油,运用十分广泛,然而其茎、叶的精油含量却相差悬殊。 为探索柠檬香茅精油代谢相关的蛋白途径,本文对柠檬香茅旗叶、成熟叶及茎秆等材料进行精油含量、总蛋白含量测定及双向凝胶电泳(2-DE)表达谱分析,运用k-means聚类分析方法对2-DE电泳中差异蛋白斑点的丰度、等电点和相对分子质量进行聚类分析和讨论,结果表明,旗叶和茎秆上调表达的蛋白质斑点的聚类对于相对分子质量变化敏感,成熟叶上调表达蛋白质斑点对于丰度的变化较为敏感。 预测了精油代谢功能相关的蛋白质斑点15个,挖取预测蛋白质斑点通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/TOF-MS)成功鉴定了9个蛋白质。 本研究为柠檬香茅精油的蛋白代谢途径提供新的基础信息及研究思路。     

Protein extraction for two-dimensional electrophoresis from olive leaf,a plant tissue containing high levels of interfering compounds

The purpose of this research is to establish a routine procedure for the application of proteomic analysis to olive tree. Olive leaf tissue is notoriously recalcitrant to common protein extraction methods due to high levels of interfering compounds. We developed a protocol for isolating proteins suitable for two-dimensional electrophoresis (2-DE) from olive leaf. The remarkable characteristics of the protocol include: (i) additional grinding dry acetone powder of leaf tissue to a finer extent, (ii) after extensive organic solvent washes to remove pigments, lipids etc., using aqueous tricholoroacetic acid washes to remove water-soluble contaminants, and (iii) phenol extraction of proteins in the presence of sodium dodecyl sulfate. The final protein preparation is free of interfering compounds based on its well-resolved 2-DE patterns. The protocol can be completed within 3 h, and protein yield is approximately 2.49 mg.g(-1) of aged leaf. We also evaluated the protocol by immunoblotting with anti-tyrosinate alpha-tubulin antibody. To our knowledge, this is the first time that a protocol for protein extraction from olive leaf appears to give satisfactory and reproducible results. The protocol is expected to be applicable to other recalcitrant plant tissues and could be of interest to laboratories involved in plant proteomics.     

Caveat receptor: proteomes on display

Display methods development is currently extending the application of this strategy beyond the generation of ligand binding reagents for research, clinical, or biotechnological purposes to its use as a primary research tool. Peptide- and cDNA display methods have the potential to contribute to understanding the mechanisms of certain classes of drugs and to help map protein-protein interactions of physiological importance. Although the critical contribution of comprehensive amino acid sequence databases has been recognized, of equal importance might be structural genomics concepts in the application of display system technology to proteomics research. Lessons learned from the study of antibody-antigen interactions are reviewed here and applied to the field of display technology with the goal of delineating major factors involved in the successful mimicry of natural protein-ligand interactions.     

Systematic analysis of the total proteins of a mammalian organism: principles, problems and implications for sequencing the human genome

High-resolution two-dimensional electrophoresis (2-DE) has reached a technological level that allows us to resolve most of the numerous unknown protein species of a mammalian organism if appropriate strategies are used. We will discuss the problems of classification and characterization of proteins and propose a systematic approach to the analysis of the total protein complex. Both a comprehensive as well as a pragmatic approach towards systematic analysis have been considered. A "complex protein database" is suggested and considered with regard to various uses. A systematic analysis of the mouse proteins has been started and some of the preliminary results are summarized here. In particular, genetic properties of the proteins were investigated and are presented in order to demonstrate the significance of a systematic analysis of proteins for research and practical application (e.g. mutagenicity testing). A concept is presented for sequencing the coding DNA of mouse and man, starting with a systematic analysis of mouse proteins and then using two recently developed methods - microsequencing of proteins from spots of 2-DE protein patterns, and utilization of the relatively short N-terminal sequences obtained - to produce the corresponding cDNA's of these proteins.